Project Summary: Most human immunodeficiency virus (HIV)-infected people, if not adherent to highly active combination antiretroviral therapy (cART), ultimately succumb to chronic infection. However, some are known as ?elite controllers? (ECs) who demonstrate superior virus control, maintaining virtually undetectable viral loads even in the absence of cART. Although HIV-specific cytotoxic T lymphocytes (CTLs) in ECs are critical for viral control and progression status, harnessing CTLs to combat HIV reservoirs remains challenging because little to no viral protein is produced in quiescent CD4+ T cells, rendering this reservoir difficult to detect by the host CTL immune response. Currently available strategies to purge latently HIV-infected cells include ?Shock and Kill,? broadly responsive TCR elicited by therapeutic vaccines, broadly neutralizing monoclonal antibodies, programmed cell death protein-1 (PD-1) blockade, and other immune checkpoint blockades. However, none of these strategies efficiently eradicates HIV reservoirs. Our preliminary data showed that strong phosphorylation of a small adaptor molecule, chicken tumor virus number 10 regulator of kinase (Crk), was induced by PD-1 signaling at the center of exhausted HIV-specific CTL immunological synapses. This critical observation prompted us to develop a novel strategy that targets a common master-signaling molecule to restore the function of exhausted CTLs to eradicate HIV reservoirs, which is an approach that is superior to targeting individual inhibitory immunoreceptor. We hypothesize that chronic HIV leads to functional impairment of CTLs via up-regulation of inhibitory receptors and that the resulting downstream phosphorylation of Crk, in turn, prevents CTL activation. We propose that exogenous phosphorylated Crk (pCrk) inhibitor could be used to restore exhausted CTL functions to eradicate HIV reservoirs. We propose the following two aims: (Aim 1) Restore the function of exhausted HIV-specific CTLs against productively infected cells by inhibiting Crk phosphorylation. pCrk inhibitors newly identified using multipronged approaches (including the state-of-the-art Alphascreen, thermal shift assay, in vitro kinase and cellular phosphoflow assay) will be tested to determine whether these pCrk inhibitors can restore functions of exhausted HIV-specific CTLs against productively infected cells. (Aim 2) Restore the function of exhausted HIV-specific CTLs against latently infected cells by inhibiting Crk phosphorylation. To further test the efficacy of pCrk inhibitors, we will determine whether pCrk inhibitors can restore the function of exhausted HIV-specific CTLs against latently infected cells. We will generate latently HIV-infected primary CD4+ T cells from peripheral blood and lymph nodes to compare their sensitivity to killing by exhausted HIV-specific CTLs with and without pCrk inhibitors. If successful, therapeutic use of small-molecule pCrk inhibitors could improve care of chronic HIV patients, restoring exhausted patient CTL defenses. Successful outcomes of this research will lead to the development of a novel immunotherapy to eradicate HIV reservoirs and associated clinical trials.